title: Recombinant expression of Laceyella sacchari thermitase in Lactococcus lactis.
abstract: Thermitase (EC 3.4.21.66) is a thermostable endo-protease with the ability to convert various food relevant substrates into low-molecular weight peptides. A thermitase produced by Laceyella sacchari strain DSM43353 was found to have a mature amino acid sequence nearly identical to that of the original thermitase isolated from Thermoactinomyces vulgaris. The DSM43353 thermitase gene sequence contains a pro-peptide including parts of an I9 inhibitor motif. Expression of the thermitase gene in the Lactococcus lactis P170 expression system allowed secretion of stable thermitase in an auto-induced fermentation setup at 30°C. Thermitase accumulated in the culture supernatant during batch fermentations and was easily activated at 50°C or by prolonged dialysis. The activation step resulted in an almost complete degradation of endogenous L. lactis host proteins present in the supernatant. Mature activated product was stable at 50°C and functional at pH values between pH 6 and pH 11, suggesting that substrate hydrolysis can be performed over a broad range of pH values. The L. lactis based P170 expression system is a simple and safe system for obtaining food compatible thermitase in the range of 100 mg/L.
authors.affiliation: Bioneer A/S, Kogle Alle 2, 2970 Hørsholm, Denmark.
authors.forename: Casper M
authors.initials: CM
authors.lastname: Jørgensen
authors.forename: Søren M
authors.initials: SM
authors.lastname: Madsen
authors.forename: Astrid
authors.initials: A
authors.lastname: Vrang
authors.forename: Ole C
authors.initials: OC
authors.lastname: Hansen
authors.forename: Mads G
authors.initials: MG
authors.lastname: Johnsen
mesh_terms: D056449:Bacillales / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C022728:thermitase
keywords: Heterologous expression; Laceyella sacchari; Lactococcus lactis; P170 Expression System; Protease; Thermitase